Apically-located P4-ATPase1-Lem1 complex internalizes phosphatidylserine and regulates motility-dependent invasion and egress in Toxoplasma gondii

The membrane asymmetry regulated by P4-ATPases is crucial for the functioning of eukaryotic cells. The underlying spatial translocation or flipping of specific lipids is usually assured by respective P4-ATPases coupled to conforming non-catalytic subunits. Our previous work has identified five P4-ATPases (TgP4-ATPase1–5) and three non-catalytic partner proteins (TgLem1–3) in the intracellular protozoan pathogen, Toxoplasma gondii. However, their flipping activity, physiological relevance and functional coupling remain unknown. Herein, we demonstrate that TgP4-ATPase1 and TgLem1 work together to translocate phosphatidylserine (PtdSer) during the lytic cycle of T. gondii. Both proteins localize in the plasma membrane at the invasive (apical) end of its acutely-infectious tachyzoite stage. The genetic knockout of P4-ATPase1 and conditional depletion of Lem1 in tachyzoites severely disrupt the asexual reproduction and translocation of PtdSer across the plasma membrane. Moreover, the phenotypic analysis of individual mutants revealed a requirement of lipid flipping for the motility, egress and invasion of tachyzoites. Not least, the proximity-dependent biotinylation and reciprocal immunoprecipitation assays demonstrated the physical interaction of P4-ATPase1 and Lem1. Our findings disclose the mechanism and significance of PtdSer flipping during the lytic cycle and identify the P4-ATPase1-Lem1 heterocomplex as a potential drug target in T. gondii.


Introduction
In eukaryotic cells, numerous subcellular pathways depend on the phospholipid composition of the membrane bilayers, which is pivotal to diverse functions, such as maintenance of the surface charge, membrane curvature and permeability, vesicular trafficking (exocytosis, endocytosis) and folding of proteins [1][2][3][4][5].
Phospholipids, comprising a significant fraction of cellular lipids, are asymmetrically present in the membranes. Phosphatidylcholine (PtdCho) and sphingomyelin are usually positioned on the exofacial leaflet, whereas phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEtn) are abundant on the cytosolic side of the plasma membrane in most cell types [2,5,6] with a few exceptions. For instance, PtdCho is dominantly located on the cytosolic leaflet of the plasmalemma and Golgi in budding yeast, but it is distributed equally between the cytosolic and luminal leaflets of other organelles. Similarly, the symmetrical distribution of PtdCho has also been seen in some mammalian cells [7]. The ATP-dependent transporters termed P4-ATPases, or flippases control this phospholipid distribution in an energy-dependent manner [8,9]. Lipid-translocating P4-ATPases (α-subunits) usually couple with a member of the non-catalytic β-subunits, which are evolutionarily conserved across eukaryotes [9][10][11][12]. The β-subunit proteins present in yeast are termed the Ligand Effector Module 3 (LEM3) or Ro-Sensitive 3 (ROS3), Cell Division Control 50 (CDC50) and transcription factor CRF1, whereas all known mammalian β-subunits belong to the CDC50 family [13][14][15]. The β-subunits assist in the folding of corresponding α-subunits (P4-ATPase), and ensure stable expression, localization and activation of P4-ATPases for lipid flipping by forming a heteromeric complex [6,11,12,[16][17][18][19].
The protozoan phylum Apicomplexa comprises over 6000 obligate intracellular parasite species of animals and humans. Toxoplasma and Plasmodium are the two exemplary pathogens of this group deployed to decipher the concepts of intracellular parasitism. In the context of this work, phospholipid synthesis and transport have been active areas of research in both parasites [20][21][22][23][24][25][26][27][28]; however, their roles in parasite signaling have emerged only recently after discovering exclusive alveolate-specific P4-ATPase-conjugated guanylate cyclase (ATPase P -GC) proteins. A series of independent studies collectively link lipid flipping with cGMP signaling and associated lifecycle events in Toxoplasma and Plasmodium species [29][30][31][32][33][34]. Two of these studies focusing on T. gondii [29] and P. yoelii [33] also disclosed a signaling complex with other interaction partners, including a LEM/CDC50 family protein. The data show the importance of the P4-ATPase domain for cGMP signaling and parasite virulence. More recently, we described five additional P4-ATPases (TgP4-ATPase1-5) and their potential β-subunits (TgLem1-3) in T. gondii [27]. The signatures of typical P4-ATPases, including ten transmembrane helices, and actuator (A), nucleotide-binding (N) and phosphorylation (P) domains, are conserved in TgP4-ATPase1-5. Not least, all except TgP4-ATPase4 are expressed during the lytic cycle of T. gondii and display varied subcellular localization in tachyzoites.
TgP4-ATPase1 resides in the plasma membrane at the (invasive) apical end of the tachyzoite stage, while TgP4-ATPase2 and TgP4-ATPase5 are present in the plasmalemma and cytomembranes. TgP4-ATPase3 is expressed in the Golgi apparatus and colocalizes with TgLem3 protein, implying their occurrence as a functional complex [27]. We used conditional mutagenesis to reveal a requisite of individual TgP4-ATPases (1−3) for the asexual growth of tachyzoites, which was corroborated in a subsequent study [35]. Meanwhile, in the malaria parasite, two distinct P4-ATPases and respective β-subunits were reported [36,37]. The ATP2 flippase of P. chabaudi, coupling with two CDC50 homologs (PcCDC50A, PcCDC50B), was found to be active in the presence of PtdSer and PtdEtn [36]. The other study demonstrated a putative P4-ATPase (ATP7) in the plasma membrane and identified CDC50C as its partner protein in P. yoelii [37]. The complex is essential for the survival of its ookinete stage in the mosquito midgut, and PtdCho was postulated as its lipid substrate. Our earlier work discovered that the acute (tachyzoite) stage of T. gondii cannot import PtdCho. However, PtdSer and PtdEtn are readily internalized from the external milieu to the parasite interior [27], and any of the P4-ATPases described above may drive the process. Herein, we demonstrate that TgP4-ATPase1 and TgLem1 form a functional heterocomplex to execute the flipping of PtdSer in the apical plasma membrane of tachyzoites. Our work discloses a physiological necessity of both proteins for the asexual growth of this clinically-relevant pathogen in human cells and signifies a novel function of PtdSer during the lytic cycle.

Genetic deletion of TgP4-ATPase1 and TgP4-ATPase2 disrupts the lytic cycle of T. gondii
We have reported the conditional mutants of TgP4-ATPase1-3 in tachyzoites, exhibiting a varying degree of growth defect upon protein depletion by indole-3-acetic acid (IAA, auxin) [27]. Knockdown of P4-ATPase3 imposed a lethal phenotype in plaque assays, while the lytic cycle of tachyzoites depleted in P4-ATPase1 and P4-ATPase2 was impaired by approximately 50 % and 80 %, respectively [27]. In this work, we engineered the deletion mutants of P4-ATPase1 and P4-ATPase2 (ΔP4-ATPase1, ΔP4-ATPase2). A dihydrofolate reductase -thymidylate synthase (DHFR-TS) selection cassette replaced the specified gene loci with the help of two sgRNAs targeting the upstream and downstream of the gene-coding region and a donor amplicon harboring 5′-and 3′-UTRs (∼1 kb) of respective P4-ATPases for double homologous recombination (Fig. 1A). The crossover events and gene deletion were verified by PCR screening of the clonal mutants using different primers (Table S1). The 5′-and 3′-recombination-specific primers yielded amplicons of the expected size (PCR1 and PCR2) in the ΔP4-ATPase1 and ΔP4-ATPase2 mutants but not in the parental strain used as a negative control (Fig. 1B). Conversely, primers binding in the coding region of P4-ATPase1 and P4-ATPase2 yielded the bands (PCR3) only in the parental strain, confirming the knockout of corresponding genes in mutants.
We examined the ΔP4-ATPase1 and ΔP4-ATPase2 mutants for their ability to form plaques, which signifies the successive lytic cycles of tachyzoites in a confluent host cell monolayer (Fig. 1C). Indeed, both mutants showed a significant growth impairment with respect to the parental strain. Quantification revealed ∼60 % and 85 % reduction in plaque area of the ΔP4-ATPase1 and ΔP4-ATPase2 strains, respectively. The effect of P4-ATPase2 deletion was more significant than the loss of P4-ATPase1. Consistent with our preceding work [27], these findings demonstrate a requirement of P4-ATPase1 and P4-ATPase2 for the lytic cycle. The fact that tachyzoites survived the deletion of individual genes and the eventual mutants could be maintained in prolonged cultures highlight unforeseen metabolic plasticity and imply a possible functional redundancy of flippases in T. gondii.

TgP4-ATPase1 functions as a phosphatidylserine flippase
P4-ATPase1 and P4-ATPase2 reside primarily in the tachyzoite's plasma membrane [27]; thus, the knockout mutants of these proteins enabled us to examine their roles in lipid translocation activity using fluorescent probes (Fig. 2). We first examined PtdSer as a potential substrate of specified flippases because it is known to be imported by tachyzoites and then further metabolized to yield PtdEtn by PtdSer decarboxylases [27,38,39]. Our assays measured the internalization of NBD-PtdSer by extracellular parasites (Fig. 2A). Before flow cytometry, the lipid-labeled parasites were mixed with propidium iodide (PI). The cell population of individual samples was analyzed based on fluorescence in green (NBD-PtdSer) and red (PI) channels following excitation by a blue laser ( Fig. 2A). As revealed by negligible PI staining, the parental control and the mutant strains displayed viability of about 90 % in samples (Fig. 2B), similar to freshly-harvested tachyzoites. Besides, most viable parental and ΔP4-ATPase2 strains exhibited a strong green signal, indicating the import of NBD-PtdSer from the milieu. In noted contrast, the ΔP4-ATPase1 strain was severely impaired in its ability to internalize the probe. Of all viable cells, only about 10 % showed a detectable NBD fluorescence, while > 90 % of the parasite population had none or weak green fluorescence (Fig. 2C). Quantifying the NBD signal in living parasites indicated a decline of > 90 % in the P4-ATPase1 mutant, whereas no change was apparent in the ΔP4-ATPase2 strain compared to the parental control (Fig. 2D).
Next, we examined the ability of both mutants to internalize NBD-conjugated PtdEtn and PtdCho. As described elsewhere [27], the PtdEtn probe was also imported by tachyzoites (Fig S1A, S1B), while no apparent internalization of NBD-PtdCho was observed in all tested strains (Fig S2A, S2B). Neither ΔP4-ATPase1 nor ΔP4-ATPase2 was defective in taking up NBD-PtdEtn, precluding their role in the flipping of PtdEtn. The flow cytometry results on lipid probes were corroborated by fluorescence microscopy (Fig S1C). The ΔP4-ATPase1 mutant was impaired in internalizing NBD-PtdSer but not the PtdEtn probe, whereas the ΔP4-ATPase2 strain behaved akin to the parental tachyzoites ( Fig. 2D, S1C). Furthermore, consistent with our preceding work [27], no signal of NBD-PtdCho was observed in any of the three strains ( Fig S2C). In conclusion, these results establish that P4-ATPase1 facilitates the flipping of PtdSer on the surface of tachyzoites.
Moreover, the apical presence of Lem1-mAID-3xHA, as verified by its colocalization with inner membrane complex marker protein TgISP1 [42] (Fig S3A), suggested its coupling with P4-ATPase1 (see below for additional results). Whereas, Lem3 localized in the Golgi network, as reported earlier [27], indicating its possible partnership with P4-ATPase3. Furthermore, Lem1 was not detectable in the budding daughter cells (Fig S3B), as judged by staining with TgIMC3, a marker of inner membrane complex during endodyogeny [43].

Depletion of TgLem1 compromises the parasite growth and internalization of PtdSer
We next tested the relative growth of the mAID-3xHA-tagged Lem1 and Lem3 mutants by plaque assays in the absence or presence of auxin (Fig. 4A). Both mutants displayed significantly reduced plaques formed in the HFF monolayers compared to the parental strain. Indeed, the average plaque size was much smaller (∼40 % of the parental control) after the depletion of Lem1 and Lem3 by IAA. The phenotype of the Lem1-mAID-3xHA mutant and apical location (Fig. 3E) implied its interaction with P4-ATPase1 and, thereby, a function in PtdSer flipping, which prompted us to test the conditional strain with NBD-PtdSer probe ( Fig. 4B-Fig. 4D), as described above ( Fig. 2A). The viability of samples was about 85 % (Fig. 4B). We observed that > 80 % of the parental and Lem1-mAID-3xHA tachyzoites could import NBD-PtdSer from their environment when cultured in the absence of IAA. While exposure to IAA did not impact the control strain, it caused a significant decline in NBD-positive cells of the Lem1 mutant (Fig. 4C). A similar observation was made when NBD fluorescence intensity associated with living tachyzoites was quantified for all samples (Fig. 4D). Overall, these results show For each strain, a vector encoding Cas9 and two sgRNAs targeting the 5′ and 3′ UTRs of the gene of interest (GoI) was co-transfected with a matching donor amplicon into tachyzoites of the RHΔku80Δhxgprt (parental) strain. The amplicon harbored a DHFR-TS selection cassette (S.C.) flanked by 5′ and 3′ crossover sequences (1 kb). The mutants were isolated after pyrimethamine selection and following clonal dilution. (B) Genomic screening of the ΔP4-ATPase1 and ΔP4-ATPase2 strains using specific primers (PCR1-3, Table S1) to demonstrate the integration of the DHFR-TS cassette at the desired locus by double homologous recombination. (C) Plaque assays using the ΔP4-ATPase1, ∆P4-ATPase2 and parental strains. Plaques formed by successive lytic cycles of tachyzoites appear as white areas on a host cell monolayer stained by crystal violet. For each strain, the representative images were analyzed to evaluate the area of 150-200 plaques from 3 assays (a. u., arbitrary units; means ± SE; ** p ≤ 0.01; **** p ≤ 0.0001).

Fig. 2. Ablation of TgATPase1 but not TgATPase2 compromises PtdSer uptake by tachyzoites. (A)
A diagram depicting the steps of lipid uptake assay. Fresh extracellular parasites were incubated with NBD-PtdSer, followed by staining with propidium iodide (PI) and eventual flow cytometry (blue laser, 488 nm). The NBD and PI signals were detected in green (530/30 nm) and red (LP670 nm) channels, respectively. (B-C) Histograms and graphs showing the distribution of tachyzoites labeled by PI (B) and NBD-PtdSer (C). To estimate the NBD-PtdSer flipping, living tachyzoites (low PI, > 90 % of the population) were analyzed in the green channel. Dead cells were excluded from the quantification. (D) NBD-PtdSer fluorescence associated with P4-ATPase1/2 mutants compared to the parental strain. Living tachyzoites were quantified irrespective of 'low' or 'high' NBD signal. Representative fluorescent images of parasites labeled with NBD-PtdSer are also shown. In panels B-D, curves signify one of the three independent assays, whereas graphs depict the means with standard error (ca. 20,000 parasites for each strain from n = 3 assays; *** p ≤ 0.001 **** p ≤ 0.0001). the importance of Lem1 for the lytic cycle and advocate its role in PtdSer flipping, possibly by coupling with P4-ATPase1.

The ΔP4-ATPase1 and Lem1-mAID-3xHA mutants phenocopy the lytic cycle defects
Impaired growth of the ΔP4-ATPase1 and Lem1-mAID-3xHA strains encouraged us to examine their physiological importance for individual steps of the lytic cycle, including replication, invasion, egress, and gliding motility (Fig. 5, S4). The parental strains corresponding to each mutant served as the controls in comparative phenotyping. To begin with, no difference in the replication of tachyzoites, as gauged by the size of parasitophorous vacuoles (number of progeny/vacuole) at 24 h and 40 h post-infection, was apparent in the mutants (Fig S4). In contrast, the loss of P4-ATPAse1 and depletion of Lem1 negatively impacted the egress and invasion at similar levels in both mutants (Fig. 5A, Fig. 5B). Notably, the egress defect was recorded only after 48 h of infection in both mutants ( Fig. 5A). All strains eventually exited with comparable efficiency in late-stage cultures (64 h infection). The ΔP4-ATPase1 and Lem1-mAID-3xHA strains were also impaired in invading host cells (Fig. 5B). Moreover, the egress and invasion defects were recapitulated in the gliding motility assays (Fig. 5C). The two mutants displayed significantly lower motile fractions and trail lengths than the respective control (parental) strains. Collectively, these findings underline the specific need for P4-ATPase1 and Lem1 for the motility-dependent egress and invasion events, although not for the intracellular replication, during the lytic cycle of T. gondii.
genomic tagging in tachyzoites, as described previously [45] (Fig. 6B). In this regard, a donor amplicon carrying BirA-3xHA and the HXGPRT cassette flanked by 5′ and 3′ homology arms of P4-ATPase1 were co-transfected along with a vector encoding Cas9 and 3′UTR-specific sgRNA into RHΔku80Δhxgprt strain. Parasites were selected with mycophenolic acid and xanthine (Fig. 6B), and positive clones harboring P4-ATPase1-BirA-3xHA fusion were confirmed by genomic screening using recombination-specific primers (Fig. 6C, Table S1). The apical presence of P4-ATPase1-BirA-3xHA shown by immunostaining excluded any transgenic artifact due to genomic tagging (Fig. 6D). Supplementation of the parasite culture with biotin allowed the BirA domain to biotinylate proteins proximal to  Fig. 2A. Histograms and graphs show the distribution of PI-and NBD-stained tachyzoites, precultured without or with indole-3-acetic acid (500 μM IAA, 24 h in HFFs). (D) NBD-PtdSer fluorescence associated with Lem1 mutant compared to the parental strain under -/+IAA conditions. Living tachyzoites irrespective of 'low' or 'high' NBD signal were quantified. In panels B-D, curves represent one of the three assays, while graphs show the mean values with standard error (ca. 20,000 parasites/strain, n = 3 assays, *** p ≤ 0.001). To calculate the NBD-PtdSer internalization, only living tachyzoites (low PI, > 90 % cells) were analyzed. P4-ATPase1 and thereby marked potential partner proteins (Fig. 6A). The parental strain in the absence or presence of biotin was included as a control.
Immunofluorescence staining of the P4-ATPase1-BirA-3xHA strain by α-HA antibody showed a robust apical signal irrespective of biotin (Fig. 6E). On the other hand, streptavidin stained the apicoplast, mitochondrion and parasitophorous vacuole membrane, corresponding to the biotin-dependent acetyl-CoA carboxylase [46], pyruvate carboxylase [47] and vacuole-associated host mitochondrial proteins, respectively. The P4-ATPase1-BirA-3xHA and parental strains were subjected to pulldown of the biotinylated proteins by streptavidin-coated magnetic beads. As expected, the α-HA-immunoblot confirmed the expression of P4-ATPase1-BirA-3xHA (∼243-kDa) (Fig. 6F). Two other bands matching with the acetyl-CoA carboxylase and pyruvate carboxylase were observed in both strains regardless of the cofactor (Fig. 6F, black triangles). Besides, immunoblot pointed to additional bands in biotin-treated P4-ATPase1-BirA-3xHA samples compared to untreated and parental controls (Fig. 6F, red triangles). The mass spectrometry analysis detected > 170 proteins, including naturally biotinylated proteins across all samples, of which only seven proteins, including P4-ATPase1 and Lem1, were identified exclusively in the P4-ATPase1-BirA-3xHA strain (Fig. 6G, Table S2). In conjunction with the above data, our proximity-dependent labeling assay strongly suggests the occurrence of a functional complex between the two proteins in tachyzoites of T. gondii.

Reciprocal immunoprecipitation endorses the physical association of P4-ATPase1 and Lem1
Encouraged by the aforementioned datasets, we set up reciprocal immunoprecipitation assays using the existing strains (P4-ATPase1-AID-3xHA [27] and Lem1-mAID-3xHA (Fig. 3)) in the presence or absence of IAA. The protein samples were analyzed by liquid chromatography-mass spectrometry (LC-MS). As shown (Fig. 6H), we detected Lem1 in α-HA-precipitated proteins of the P4-ATPase1-AID-3xHA strain cultured in the absence of IAA (on-state, P4-ATPase1replete) but not in the presence (off-state, P4-ATPase-depleted). Similarly, we found P4-ATPase1 in samples after α-HA-pulldown of the Lem1-mAID-3xHA strain incubated without IAA (Lem1-replete) but not when exposed to the compound (Lem1-depleted). As expected, the bait proteins were detected in both mutants (Fig. 6H). These experiments provide strong evidence of physical interaction between the two proteins for PtdSer translocation during the lytic cycle of T. gondii.

Discussion
Our prior work has demonstrated the translocation of PtdSer and PtdEtn into tachyzoites, identified five P4-ATPases and three LEM/ CDC50-family proteins and disclosed the physiological importance of P4-ATPase1-3 for the lytic cycle of T. gondii [27]. Here, we show the P4-ATPase1-Lem1 complex, located at the invasive (apical) pole of tachyzoites, as a significant player in PtdSer flipping and motilitydependent egress and invasion (Fig. 7). Besides, this study reveals that P4-ATPase1 and P4-ATPase2 are required for a normal lytic cycle but dispensable for the survival of tachyzoites. Both proteins are considered to be essential according to a genome-wide phenotypic screening [40] and based on conditional mutagenesis in a recent study [35], overlapping with our previous report [27] and this work. We demonstrate that tachyzoites can cope with the individual deletion of P4-ATPase1 and P4-ATPase2. The corresponding mutants continue to grow in prolonged cultures, underlining the unexpected and previously-unknown plasticity of phospholipid flipping in T. gondii. Any functional redundancy and synthetic lethality of P4-ATPases warrant combinative mutagenesis in tachyzoites. and gliding motility (C) using standard immunostaining methods. About 1000 parasites of each strain were examined to estimate the invasion efficiency. The natural egress of tachyzoites was measured by scoring 500-600 vacuoles/strain (n = 3 assays). Where indicated, IAA was added 24 h post-infection. For gliding motility, immunostained tachyzoites from three experiments were evaluated for the motile fraction (500 parasites/strain) and trail lengths (100-120 trails/strain). Tachyzoites were precultured for 24 h without or with IAA before the invasion and motility assays were set up. Graphs in panels A-C show data from 3 assays (means ± SE, ** p ≤ 0.01, *** p ≤ 0.001).
Most orthologs of apicomplexan P4-ATPases in mammalian cells and yeast are known to interact with a non-catalytic β-subunit (LEM/ CDC50) for their flippase function. Similarly, we show that Lem1 and Lem3, located in the apical region and the Golgi network, respectively, are crucial for the parasite growth; and the knockdown of the former protein phenocopies the P4-ATPase1 mutant (Fig. 7). Our work shows the interaction of P4-ATPase1 with Lem1 and suggests coupling of P4-ATPase3 with Lem3, likely in the form of a functional heterocomplex. As LEM/CDC50 proteins can interact with more than one α-subunits [51], hence it is plausible that TgLem1 may also associate with other P4-ATPases and thereby contribute to the translocation of other phospholipids in tachyzoites. Besides Lem1, we found a few other proteins interacting with P4-ATPase1, including a phosphatidylinositol 3/4-kinase, which remained to be examined in the future.
The endocytic-like structures have been described and known to internalize lipids in intracellular tachyzoites [52]. Likewise, lipid probes can also be endocytosed by extracellular parasites, although trafficking factors remain poorly understood [53]. Thus, we cannot rule out the role of endocytosis in phospholipid internalization; however, the observed defect in translocation of PtdSer but not of PtdEtn in the ΔP4-ATPase1 mutant and selectivity of lipid uptake in tachyzoites demonstrate the functional importance of P4-ATPase1-Lem1 heterocomplex in T. gondii. Accordingly, reduction in the motility, invasion and egress in both ΔP4-ATPase1 and Lem1-mAID-3xHA mutants point to a role of the P4-ATPase1-Lem1 complex and PtdSer in exocytosis of secretory organelles [35]. Whether and how PtdSer, PtdThr and/or any other lipid regulate the observed phenotype remains to be examined. In conclusion, the shared apical location, flipping activity, phenotypic traits and physical association strongly advocate a functional coupling of P4-ATpase1 and Lem1 as α-and β-subunits in tachyzoites. Besides, a physiological requirement of both proteins underscores the therapeutic potential of PtdSer flipping during acute toxoplasmosis.

Biological resources and reagents
Human foreskin fibroblast (HFF) cells to maintain tachyzoites of T. gondii were provided by Carsten Lüder (George-August University, Göttingen). The RHΔku80Δhxgprt strain lacking hypoxanthine-xanthine-guanine phosphoribosyltransferase (Δhxgprt) as well as the nonhomologous end-joining DNA repair (Δku80) was offered by Vern Carruthers (University of Michigan, USA) [54][55][56]. The RHΔku80Δhxgprt-TIR1 strain and the plasmids (pLinker-mAID-3xHA-HXGPRT, pLinker-BirA-3xHA-HXGPRT) were gifted by David Sibley (Washington University, USA) [41]. The pUC19 vector for making the gene-specific donor amplicons for 5′ and 3′ recombination was provided by Bang Shen (Huazhong Agricultural University, Wuhan China) [57]. The primary antibodies binding TgGap45 and TgHsp90 proteins were provided by Dominique Soldati-Favre (University of Geneva, Switzerland) and Sergio Angel (IIB-INTECH, Buenos Aires, Argentina), respectively. Antibodies against TgISP1 and TgIMC3 were offered by Peter Bradley (University of California, Los Angeles, USA) and Marc-Jan Gubbels (Boston College, MA, USA). Other primary antibodies recognizing the HA epitope and TgSag1 were procured from Takara-Bio (Japan) and Sigma-Aldrich (Germany). The secondary antibodies (Alexa488, Alexa594; IRDye 680RD, 800CW) and oligonucleotides (Table S1) were obtained from ThermoFisher Scientific (Germany). The cell culture media and additives were purchased from PAN Biotech (Germany), and other standard chemicals were supplied by Sigma-Aldrich and Carl Roth (Germany). The reagent kits for isolation, cloning and purification of nucleic acids were acquired from Analytik Jena and Life Technologies (Germany). Lipids tagged with C6-nitrobenzoxadiazole (NBD) were acquired from Avanti Polar Lipids (USA).

Generation of P4-ATPase knockout mutants
The P4-ATPase genes were replaced by a dihydrofolate reductasethymidylate synthase (DHFR-TS) selection cassette. The process of double homologous recombination at the targeted locus was facilitated by a dual-CRISPR-Cas9 plasmid expressing Cas9 and two sgRNAs binding to the 5′ UTR and 3′ UTR regions of respective P4-ATPase genes. The vector was generated by Q5 site-directed mutagenesis kit (New England Biolabs). The 5′ UTR (1 kb before the start codon) and 3′ UTR (1 kb after the stop codon) of P4-ATPase1 and P4- ATPase2 were amplified from the genomic DNA and cloned into the pUC19 vector flanking the DHFR-TS selection cassette using a Gibson assembly kit (Vazyme Biotech). The locus-specific donor amplicons were generated from the pUC19 constructs and co-transfected along with the matching CRISPR-Cas9 vectors encoding 5′sgRNA and 3′sgRNA for P4-ATPases into the RHΔku80Δhxgprt strain. Tachyzoites expressing DHFR-TS were selected by 1 μM pyrimethamine [58] and cloned by limiting dilution in 96-well plates. The knockout mutants were screened by crossover-specific genomic PCR.

Parasite phenotyping assays
Plaque assays were performed to examine the overall fitness of the mutants in confluent HFF cells. Host cells were infected (200 tachyzoites/well) and incubated for seven days under standard culture conditions without any perturbation. Samples were fixed with ice-cold methanol (−80 °C, 10 min) and stained with crystal violet dye for 15 min, followed by PBS washing. Plaques were imaged, and their size/area was measured using Adobe Photoshop (2020 suite). To quantify the replication rate, HFFs grown on coverslips were infected (10 5 parasites, 24 h and 40 h), followed by fixation, neutralization, permeabilization, blocking and staining with the α-TgGap45 and Alexa594 antibodies (see above). The cell division was scored by counting parasites developing within their parasitophorous vacuoles.
For the motility assay, tachyzoites (4 x 10 5 ) were suspended in calcium-and magnesium-free Hank's balanced salt solution (HBSS) and centrifuged (300 g, 5 min, 37 °C) to settle them on the BSAcoated (0.01 % in PBS) coverslips in a 24-well plate. Samples were incubated in a humidified incubator (15 min, 37 °C), followed by fixation in 4 % paraformaldehyde/PBS (15 min) and neutralization with 0.1 M glycine/PBS (5 min). Samples were blocked in 3 % BSA/PBS (30 min) and immunostained by mouse α-TgSag1 (1:200) and Alexa488 antibodies prepared in the blocking buffer (1 h). The fraction of moving parasites was counted by a microscope, whereas the average trail lengths were quantified using the ImageJ program.

Proteolytic digestion
Samples were processed by single-pot solid-phase-enhanced sample preparation (SP3) [61,62]. In brief, biotinylated proteins were released from the streptavidin-coated beads by incubation for 5 min at 95 °C in an SDS/biotin-containing buffer (1 % (w/v) SDS, 10 mM biotin, 10 mM TRIS, pH 7.5). The eluted proteins were reduced and alkylated by dithiothreitol and iodoacetamide. Subsequently, 2 μL of carboxylate-modified paramagnetic beads were added to samples (Sera-Mag SpeedBeads, GE Healthcare, 0.5 μg solids/μL in water, [61]). After adding acetonitrile to a concentration of 70 % (v/v), samples were allowed to settle at room temperature for 20 min. Beads were washed twice in water with 70 % (v/v) ethanol and then once with acetonitrile. They were resuspended in 50 mM NH 4 HCO 3 containing trypsin (Promega) at an enzyme-to-protein ratio of 1:25 (w/w) and incubated overnight at 37 °C. After proteolytic digestion, acetonitrile was added to a concentration of 95 % (v/v), followed by incubation at room temperature (20 min). To increase the yield, supernatants derived from this initial peptide-binding step were subjected to the SP3 peptide purification procedure [62]. Samples were washed with acetonitrile. To recover the peptides, paramagnetic beads from the original sample and corresponding supernatants were pooled in 2% (v/v) dimethyl sulfoxide, sonicated for 1 min and then centrifuged (12,500g, 2 min, 4 °C). Supernatants containing tryptic peptides were transferred into a glass vial for mass spectrometry.

Liquid chromatography-mass spectrometry (LC-MS) analysis
Tryptic peptides were separated using an Ultimate 3000 RSLCnano LC system equipped with a PEPMAP100 C18 5 µm 0.3 × 5 mm trap (ThermoFisher Scientific) and an HSS-T3 C18 1.8 µm, 75 µm × 250 mm analytical reversed-phase column (Waters Corporation). Mobile phase A was water containing 0.1 % (v/v) formic acid and 3 % (v/v) DMSO. Peptides were separated running a gradient of 2-35 % mobile phase B (0.1 % (v/v) formic acid, 3 % (v/v) DMSO in acetonitrile) for 40 min at a flow rate of 300 nL/min. The total analysis time was 60 min, including wash and column re-equilibration steps. The column temperature was set to 55 °C. Mass spectrometric analysis of eluting peptides was conducted on an Orbitrap Exploris 480 instrument (ThermoFisher Scientific). The spray voltage was set to 1.8 kV, the funnel RF level to 40, and the heated capillary temperature was kept at 275 °C.
Data were acquired either in data-dependent acquisition (DDA) or in data-independent acquisition (DIA) mode. For DDA, the ten most abundant peptides (Top10) were targeted for fragmentation. Full MS1 resolution was set to 120,000 at m/z 200, and the automated gain control (AGC) target to 300 % with a maximum injection time of 50 ms. The mass range was set to m/z 350-1500. For MS2 scans, the collection of isolated peptide precursors was limited by an ion target of 1 × 10 5 (AGC target value of 100 %) and maximum injection times of 25 ms. Fragment ion spectra were acquired at a resolution of 15,000 at m/z 200. The intensity threshold was adjusted at 1E4. The isolation window width of the quadrupole was set to m/z 1.6, and the normalized collision energy was fixed at 30 %. In DIA mode, full MS1 resolution was set to 120,000 at m/z 200 and AGC target to 300 %. The mass range was set to m/z 345-1250. Fragment ion spectra were acquired with an AGC target value of 1000 %, applying a DIA scheme consisting of 21 windows with variable width and a 0.5 Th overlap. The resolution was set to 30,000, and ion transfer time was determined in "auto mode". The normalized collision energy was fixed at 27 %. Data were acquired in profile mode using positive polarity.

Data analysis and label-free quantification
The DDA-derived raw data acquired with the Exploris 480 were processed with MaxQuant (v2.0.1) [63,64] using standard setting and label-free quantification (LFQ) enabled for each parameter group, i.e., P4-ATPase1-BirA-3xHA and parental samples (LFQ min ratio count 2, stabilize large LFQ ratios disabled, match-betweenruns). Data were searched against the forward and reverse sequences of the T. gondii proteome (UniProtKB/TrEMBL, 8450 entries, UP000005641, released November 2021) and a list of common contaminants. For peptide identification, trypsin was set as a protease, allowing for two missed cleavages. Carbamidomethylation was set as a fixed and oxidation of methionine and acetylation of Ntermini as variable modifications. Only peptides with a minimum length of 7 amino acids were considered. Peptide and protein false discovery rates (FDR) were set to 1 %. In addition, proteins were identified based on at least two peptides. Statistical analysis was conducted using the Student's t-test, corrected by the Benjamini-Hochberg (BH) method for multiple hypothesis testing (FDR, 0.01). Only proteins over two-fold enrichment compared to the controls were selected.
The DIA-derived raw data acquired with the Exploris 480 were processed using DIA-NN (v1.8.0.1), applying the default parameters for library-free database search. Data were analyzed using a custom-compiled database containing UniProtKB/TrEMBL entries of the T. gondii proteome and common contaminants. For peptide identification and in-silico library generation, trypsin was set as a protease, allowing for one missed cleavage. Carbamidomethylation was set as a fixed modification, and the maximum number of variable modifications was set to zero. The peptide length ranged from 7 to 30 amino acids. The precursor m/z range was set to 300-1800, and the product ion m/z range to 200-1800. For quantification, we used the robust LC (high precision) mode. Cross-run normalization was disabled ("off"). We applied the in-built algorithm of DIA-NN to automatically optimize MS2 and MS1 mass accuracies and scan window size. Peptide precursor FDRs were controlled below 1 %.

Data presentation and statistics
Unless stated otherwise, all assays shown in this study were done at least three independent times. Figures illustrating images or making of transgenic strains typically show only a representative of three or more biological assays. Graphs and statistical significance were generated using GraphPad Prism (v8). The error bars in graphs signify means with S.E. from multiple assays. The p-values were calculated by Student's t-test (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001).

CRediT authorship contribution statement
NG conceived, coordinated and supervised the work; NG and KC designed the study; KC and XH performed experiments and generated data; UD and ST carried out the proteomics study; KC, ÖGE and NG analyzed results and drafted the manuscript. All authors reviewed and approved the work.